[The Mechanism of Bimodal Effect of DL-Butyonine Sulfoximine on Constitutive Androstane and Pregnane X Receptors In Vitro].

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are nuclear receptors that are involved in the regulation of gene transcription of enzymes that are responsible for biotransformation and excretion of endo- and xenobiotics. The goal of the work was to study the effect of DL-butyonine sulfoximine (BSO, gamma-glutamylcysteine synthetase inhibitor) on the relative amounts of CAR and PXR in Caco-2 cells and to clarify its mechanisms. BSO was used at concentrations of 1-500 µM for 24 and 72 h. The generation of reactive oxygen species (ROS) has been evaluated using the MitoTracker Red CM-H2 XRos fluorescent probes. Cytotoxicity was analyzed by the MTT test. The relative amount of CAR and PXR was assessed by the Western blot method. It has been shown that BSO caused an increase in ROS formation at concentrations of 10, 50, and 100 µM for 24 h and at concentrations of 50 and 100 µM for 72 h. However, 500 µM BSO reduced the viability of cells during all periods of exposure. The relative amount of CAR increased in 24 h at the BSO concentrations of 50 and 100 µM and in 72 h at its concentrations of 10 and 50 µM. The amount of PXR increased in 72 h during incubation with BSO at the concentration of 50 µM and in 24 and 72 h at its concentrations of 100 and 500 µM. The combined use of BSO (50 µM, 24 h; 10 and 50 µM, 72 h) and glutathione inhibited CAR induction, whereas 50 and 100 µM BSO inhibited PXR formation for 72 h. The addition of 1 mM glutathione to the nutrient medium with BSO (100 and 500 µM, 24 h; 500 µM, 72 h) did not affect the relative amount of PXR. No effect on CAR was observed when 1 mM glutathione was used together with BSO (100 µM, 24 h; 50 and 100 µM, 72 h). Thus, BSO can induce CAR and PXR formation by both increasing the production of free radicals, thus developing oxidative stress, and by acting independently as a xenobiotic.

[Functioning of pregnan X receptor under conditions of nitrosative stress]. / Funktsionirovanie pregnan X retseptora v usloviiakh nitrozativnogo stressa.

Pregnan X receptor (PXR) is a nuclear receptor that plays an important role in the regulation of the expression of biotransformation and metabolic enzymes. The functioning and possible mechanisms of PXR regulation under conditions of nitrosative stress have not been studied, which served as the purpose of this study. The work was performed on Caco-2 cells. Nitrosative stress (NS) was modeled using S-nitrosoglutathione (GSNO) at concentrations of 1 µM, 10 µM, 50 µM, 100 µM, and 500 µM and incubation during of 3 h, 24 h, and 72 h. The amount of PXR was assessed byWestern blotting. Incubation of Caco-2 cells with all concentrations GSNO for 3 h led to a decrease in the amount of PXR. Incubation with GSNO (1-50 µM) for 24 h was accompanied by an increase in the amount of PXR, while at a concentration of 100 µM this indicator did not significantly differ from the control, at a concentration of 500 µM it was lower. Prolonged incubation (72 h) enhanced NS and led to a normalization (1 µM GSNO) or a decrease of the PXR level (10-500 µM GSNO). The induction of PXR by GSNO was mediated by the effect of the nitrosative stress product bityrosine on the transcription factor. It was shown that bityrosine at concentrations of 0,4 mM and 1 mM increased the amount of PXR.

Induction of Constitutive Androstane Receptor during the Development of Oxidative Stress.

We studied the effect of 3-, 24-, and 72-h exposure to H2O2 in concentrations of 0.1-100.0 µM on the level of constitutive androstane receptor in Caco-2 cells. It was shown that 3- and 24-h incubation with Н2О2 in all concentrations had no effect on the level of constitutive androstane receptors. Increasing the incubation time to 72 h led to an increase in the level of constitutive androstane receptor at H2O2 concentrations of 5, 10, and 50 µM and to a decrease at a concentration of 100 µM. Antioxidant glutathione (1 mM) in parallel to the prooxidant neutralized these changes.